Optical microscopy-based approaches to the detection and analysis of particles of micron and sub-micron dimensions, suspended or otherwise present within fluid samples, encounter several practical challenges. One is how to provide a clean, sealed environment to contain the fluid of interest. This is typically addressed by placing the fluid either in the well of a microscope slide, under a cover glass, or within immediate contact with the objective lens, scanning probe (e.g. scanning, tunneling, atomic force, near-field) microscopy and the like. Such solutions are limited by a field of view <100 um in diameter and scanning rates typically measured in minutes.
Similar techniques involve a chamber formed into an optical disc, where it can be “read” when the disc is inserted into an optical disc player. Such optical-disc/player based systems in the prior art lack the necessary provisions to reliably focus and track the surface, calibrate the optical system, and to insert, filter, contain, sort, analyze, and dispose of fluids and potential biohazards.
Another challenge to any approach that uses conventional optical systems is that spatial resolution is limited by diffraction effects, to approximately the wavelength of the optical beam. Many particles of interest—for example microvesicles in biological samples—have dimensions well below such “diffraction limits”, and so they cannot be observed using conventional optical imaging systems. One alternative is to use more complex scanning systems, that employ a combination of optical and atomic force microscopy. These use AFM probes, micro-cantilevers for example, to in effect create a very small aperture through which near-field imaging is carried out. Such systems require the sample to be positioned extremely close to the optical elements capturing the images, and are relatively complex in operation, as well as expensive. They also share the problem mentioned above of limited field of view and scanning rate limitations.
There remains, therefore, a need for systems and methods that not only provide a clean, sealed environment, necessary for the detection and analysis of fluid samples, but that interrogate the samples using a reasonably simple optical system characterized by a large effective field of view. This may be achieved by an appropriate choice of scanning rate for a sub-micron optical stylus, which could, for example, allow for the formation of a 3-dimensional image approximately 100 mm in diameter within 2 hours. Ideally, the optical system would also provide “super” spatial resolution, significantly smaller than the wavelength of the optical beam, allowing particles with dimensions of a few hundreds or even tens of nanometers to be imaged.